Somatostatin Analogs
Somatostatin is a cyclic tetradecapeptide found both in the central nervous system and in peripheral tissues. It was originally isolated from mammalian hypothalamus and identified as an important inhibitor of growth hormone secretion from the anterior pituitary. Its multiple biological activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut hormones and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central nervous system (for review see Lamberts, Endocrine Rev., 9:427, 1988). Additionally, somatostatin and its analogs are potentially useful antiproliferative agents for the treatment of various types of tumors.
Natural somatostatin (also known as Somatotropin Release Inhibiting Factor, SRIF) of the following structure:H-Ala1-Gly2-Cys3-Lys4-Asn5-Phe6-Phe7-Trp8-Lys9-Thr10-Phe11-Thr12-Ser13-Cys14-OH  (SEQ ID NO:1)                was first isolated by Guillemin and colleagues (Bruzeau et al. Science, 179:78, 1973). It exerts its effect by interacting with a family of receptors. Recently, five receptor subtypes, termed SSTR1 to 5, have been identified and cloned. The precise functional distinction between these receptor subtypes has not yet been fully elucidated.        
In its natural form, somatostatin has limited use as a therapeutic agent since it exhibits two undesirable properties: poor bioavailability and short duration of action. For this reason, great efforts have been made during the last two decades to find somatostatin analogs that will have superiority in either potency, biostability, duration of action or selectivity with regard to inhibition of the release of growth hormone, insulin or glucagon.
Structure-activity relation studies, spectroscopic techniques such as circular dichroism and nuclear magnetic resonance, and molecular modeling approaches reveal the following: the conformation of the cyclic part of natural somatostatin is most likely to be an antiparallel β-sheet; Phe6 and Phe11 play an important role in stabilizing the pharmacophore conformation through hydrophobic interactions between the two aromatic rings; the four amino acids Phe7-Trp9-Lys9-Thr10 which are spread around the β-turn in the antiparallel β-sheet are essential for the pharmacophore; and (D)Trp8 is preferable to (L)Trp8 for the interactions with somatostatin receptor subtypes 2 through 5.
Nevertheless, a hexapeptide somatostatin analog containing these four amino acids anchored by a disulfide bridge: is almost inactive both in vitro and in vivo, although it has the advantage of the covalent disulfide bridge which replaces the Phe sup 6-Phe sup. 11 hydrophobic interactions in natural somatostatin.
Four main approaches have been attempted in order to increase the activity of this hexapeptide somatostatin analog. (1) Replacing the disulfide bridge by a cyclization which encourages a cis-amide bond, or by performing a second cyclization to the molecule yielding a bicyclic analog. In both cases the resultant analog has a reduced number of conformational degrees of freedom. (2) Replacing the original residues in the sequence Phe7-(D)Trp8-Lys9-Thr10 with other natural or non-natural amino acids, such as replacing Phe7 with Tyr7 and Thr10 with Val10. (3) Incorporating additional functional groups from natural somatostatin with the intention that these new elements will contribute to the interaction with the receptor. (4) Eliminating one of the four amino acids Phe7-(D)Trp8-Lys9-Thr10 with the assumption that such analogs would be more selective.
The somatostatin analog, MK-678:cyclo(N-Me-Ala6-Tyr7-(D)Trp8-Lys9-Val10-Phe)  (SEQ ID NO:3)is an example of a highly potent somatostatin analog designed using the first three approaches above (Veber, et al., Life Science, 34:371, 1984). In this hexapeptide analog, a cis-amide bond is located between N-Me-Ala and Phe11, Tyr7 and Val10 replace Phe7 and Thr10 respectively, and Phe11 is incorporated from natural somatostatin.
Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and 4,235,886) includes Octreotide: the first approved somatostatin analog clinically available and it was developed using the third approach described above. Here, (D)Phe5 and the reduced C-terminal Thr12-CH2OH are assumed to occupy some of the conformational space available to the natural Phe6 and Thr12, respectively.
The compound TT-232. is closely related to Octreotide and is an example of implementing the fourth approach described above. The lack of Thr10 is probably responsible for its high functional selectivity in terms of antitumor activity.
These examples of highly potent somatostatin analogs suggest that the phenylalanines in positions 6 and 11 not only play an important role in stabilizing the pharmacophore conformation but also have a functional role in the interaction with the receptor. It is still an open question whether one phenylalanine (either Phe6 or Phe11) is sufficient for the interaction with the receptor or whether both are needed.
It is now known that the somatostatin receptors constitute a family of five different receptor subtypes (Bell and Reisine, Trends Neurosci., 16, 34-38, 1993), which may be distinguished on the basis of their tissue specificity and/or biological activity.
Therapeutic Uses of Somatostatin Analogs
By virtues of their inhibitory pharmacological properties, somatostatin analogs can be used for the treatment of patients with hormone-secreting and hormone-dependent tumors. At the present, symptoms associated with metastatic carcinoid tumors (flushing, diarrhea, valvular heart disease, and abdominal pain) and vasoactive intestinal peptide (VIP) secreting adenomas (watery diarrhea) are treated with Octreotide. Octreotide has also been approved for the treatment of severe gastrointestinal hemorrhages and Acromegaly. In addition, the abundance of high affinity somatostatin receptors in various tumors enables the use of radio-label somatostatin analogs in-vivo for visualization of these tumors (Lamberts et al. N. Engl. J. Med., 334:246 1996). In neuroendocrine tumors, particularly Carcinoids and VIPomas, Oceotide inhibits both the secretion and the effect of the active agent. Thus, in VIPomas characterized by profuse secretory diarrhea, Somatostatin analogs reduce the diarrhea through the inhibition of VIP secretion, and by direct effect on intestinal secretion. However, response to the drug often decreases with time, possibly due to down-regulation of somatostatin receptors on tumor cells or to the generation of receptor negative clone. The absence of consistent antiproliferative effect may be related to the poor affinity of Octeotide to some of the somatostatin receptor subtypes found in these tumors (Lamberts et al. Ibid.).
Native somatostatin and Octreotide reportedly improve secretory diarrhea symptoms, other than those associated with neuroendocrine tumors. Control of secretory diarrhea associated with short bowel syndrome, ileostomy diarrhea, idiopathic secretory diarrhea associated with amyloidosis, and diabetic diarrhea have been reported. Both compounds have also shown some promise in the management of refractory diarrhea related to AIDS, especially in patients without identifiable pathogens. Somatostatin analogs known in the art may not provide sufficient selectivity or receptor subtype selectivity, particularly as anti-neoplastic agents (reubi and Laissue, TIPS, 16, 110-115, 1995).
Somatostatin analogs selective to type 2 and 5 receptors which inhibit growth hormone but not insulin release may potentially be used for treatment of Non Insulin Dependent Diabetes Mellitus (NIDDM). Lower potency on glucagon-release inhibition is preferred for reduction of peripheral resistance to insulin and improvement of glycemic-control.
Growth hormone is a direct antagonist of the insulin receptor in the periphery and growth hormone overproduction is associated with insulin peripheral resistance. Elevated IGF, which is the principal biological signal of growth hormone, is associated with diabetic complications such as angiopathy, retinopathy, and nephropathy. Nephropathy is one of the major complications of diabetic angiopathy and one of the leading causes of end stage renal failure and death in diabetic patients. Evidence of the significant involvement of the GH-IGF axis in diabetic and other nephropathies has been provided by several studies (Flyvbjerg A. Kidney Int. S12-S19, 1997). It was recently found that increased serum growth hormone levels in the Non-Obese-Diabetic (NOD) mice are similiar to the changes described in humans (Landau et al., J. Am. Soc. Nephrol. 8:A2990, 1997). These finding enable the slucidation of the role of the growth hormone-IGF axis in diabetic retinopathy and testing somatostatin analogs for potentially therapeutic effect in these secondary diabetes-associated complications.
Improved Peptide Analogs
It would be desirable to achieve peptide analogs with greater specificity to receptor subtypes therby achieving enhanced clinical selectivity.
As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical utilities. Thus in the last few years new methods have been established for the treatment and therapy of illnesses in which peptides have been implicated. However, the use of peptides as drugs is limited by the following factors: a) their low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; b) their poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; c) their rapid excretion through the liver and kidneys; and d) their undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism.
It would be most beneficial to produce conformationally constrained peptide analogs overcoming the drawbacks of the native peptide molecules, thereby providing improved therapeutic properties.
A novel conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., (Bio-polymers 31:745, 1991) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. While the concept was envisaged as being applicable to any linear peptide of interest, in point of fact the limiting factor in the proposed scheme was the availability of suitable building units that must be used to replace the amino acids that are to be linked via bridging groups. The actual reduction to practice of this concept of backbone cyclization was prevented by the inability to devise any practical method of preparing building units of amino acids other than glycine (Gilon et al., J. Org. Chem., 587:5687 1992).
Further disclosures by Gilon and coworkers (WO 95/33765 and WO 97/09344) provided methods for producing building units required in the synthesis of backbone cyclized peptide analogs. Recently, the successful use of these methods to produce backbone cyclized peptide analogs having somatostatin activity was also disclosed (WO 98/04583). All of these methods are incorporated herein in their entirety, by reference.
None of the background art teaches or suggest the somatostatin analogs disclosed herein having improved therapeutic selectivity.